Purifying DNA from tissue or cell samples is complicated, time consuming, and often requires chemical and equipment that are hazardous and/or expensive. Most current methods for DNA preparation use traditional organic solvent extractions and/or absorption columns. In general, optimal recovery of DNA from biological samples is achieved by a phenol extraction followed by ethanol precipitation. This requires training and technical skills so that DNA is obtained substantially free of proteins and RNA.
Clinically useful applications of DNA purification from human tissues, for example, involve the detection of disease-causing, viral-specific genomes incorporated into human chromosomes, such as human immunodeficiency virus (HIV). Another useful application is the detection of disease causing genes, such as cystic fibrosis, sickle cell anemia, and Duchenne muscular dystrophy.
Kits are now available which allow DNA isolation from tissues without the use of phenol/chloroform extraction. For example, A.S.A.P..TM. Genomic DNA Isolation Kit, Boehringer Mannheim Biochemicals, Indianapolis, Ind.; and The Extractor.TM., Molecular Biosystems, Inc., San Diego, Calif. These kits employ an ion exchange column to retain DNA on the basis of DNA's electrical charge. A disadvantage of these kits is that the entire procedure from cell lysis to elution of purified DNA requires two to four hours for most samples.
Known procedures for DNA purification from whole blood require cumbersome and time-consuming steps for cell lysate preparation. A typical method for preparing specimens of whole blood to purify DNA involves first purifying the mononuclear cells by banding in a density gradient such as ficoll hypaque (Pharmacia, Inc.) washing, then lysing the cells. The cell purification step is necessary because hemoglobin is reported to interfere with the PCR amplification. The isolated mononuclear cells are washed twice with phosphate buffered sales (PBS), then resuspended in 1 ml of PBS. A smear of the cell suspension is made and stained with Wright stain. The proportion of mononuclear cells is consistently found to be greater than 95%. A white cell count is then determined in a Coulter counter on an aliquot of each cell suspension. The cells are then pelleted and lysed by a quick lysis method to give a minimum cell concentration of 3.times.10.sup.6 cells per ml. Proteinase K is added to a final concentration of 120 micrograms/ml and the lysates are incubated at 60.degree. for 1 hour. The proteinase K is then inactivated by a 10-minute incubation at 95.degree. C..
Mass screening of the human blood supply would require a mass scale-up of a traditional DNA purification method to detect viral-specific DNA. The cost would be very high for scaling up these methods to purify DNA obtained from either large numbers of samples or large sample volumes collected from a large portion of the population. Accordingly, it is desirable to have a method for rapidly, simply, and inexpensively obtaining purified DNA from small or large volumes or numbers of samples of donated human blood or other tissues.
Furthermore, it would be beneficial if such a simple procedure suitable for rapidly purifying DNA yielded the DNA substantially free of contaminants that can interfere with hybridization techniques or the polymerase chain reaction. Such contaminants include RNA, heparin, detergents, and large amounts of some proteins, like hemoglobin. it would be further desirable for this simple method to yield large amounts of DNA that can be examined for the presence of a single copy of a targeted sequence using the PCR.
Therefore, there is a need for a convenient and reliable technique for purifying large amounts of DNA from biological tissue or cell samples that requires less time than current techniques and does not require organic, hazardous, or expensive reagents. A technique is also needed which can be inexpensively and easily scaled up or down, does not require prior separation of cells from the sample, such as red blood cells, and that yields purified DNA that is substantially free of RNA, proteins, and other contaminants interfering with detection of specific DNA sequences by hybridization and amplification techniques, including polymerase chain reaction techniques.